A large body of data has demonstrated that aberrant expression of VEGF (Vascular Endothelial Growth Factor) plays a critical role in the abnormal vascularisation associated with several human diseases. Angiogenic responses stimulated by VEGF involve the activation of the calcineurin/NFAT (Nuclear Factor of Activated T-cells) signal transduction pathway. We have previously identified an inhibitory interaction between the Plasma Membrane Calcium ATPase (PMCA) family of proteins and calcineurin in HEK-293 and breast cancer cells. PMCAs are enzymatic low capacity, high-affinity systems involved in the extrusion of Ca2+ from the cell. It is thought that PMCA regulates the activity of the calcineurin/NFAT pathway by tethering calcineurin to low Ca2+ microdomains created by the Ca2+ extrusion activity of the pump. Given the relevance of the calcineurin/NFAT pathway in the progression of angiogenesis, we have investigated whether PMCA plays a significant role as an endogenous inhibitor of VEGF-mediated angiogenesis. “Gain of function” studies conducted in HUVEC cells overexpressing PMCA4 by infection with Ad-PMCA4 (an adenovirus encoding human PMCA4), resulted in a significant decrease in the VEGF-induced activation of the calcineurin/NFAT pathway (31.92±8.74% reduction compared to the value observed in cells infected with control adenovirus Ad-LacZ, n=5). Similarly, VEGF-induced up-regulation of the NFAT-dependent, angiogenic genes Cox-2 and RCAN-1 was significantly attenuated (55.04±4.12%, n=5, and 48.42±4.72%, n=5, decrease respectively) by infection with Ad-PMCA4. PMCA4 overexpression also reduced the migration (39.8±4.08 reduction, n=6) and in vitro tube formation (42.24±10.92 reduction, n=5) of VEGF-stimulated HUVEC cells infected with Ad-PMCA4b. Moreover, in vivo angiogenesis assays performed by subcutaneous inoculation of a mixture of low growth factor-matrigel, VEGF and adenoviral vectors, in mice anesthetized with an intra-peritoneal injection of ketamine (75mg/kg)-xylazine (10mg/kg) showed a reduction of blood vessel formation (41.8±8.55 decrease, n=8) in plugs containing Ad-PMCA4. Conversely, “loss of function” experiments performed in mouse lung endothelial cells (MLEC) isolated from PMCA4-/- knock-out animals showed a significant increase in the activation of the calcineurin/NFAT pathway (4.25±1.08 fold-increment), the expression of the RCAN-1 protein as observed by western blot (n=3) and the migration of cells in wound-healing assays (1.66±0.56 fold-increment) when compared to the corresponding values in wild-type MLEC cells These results strongly demonstrate that PMCA4 plays a critical role as a negative regulator of VEGF-mediated angiogenesis via inhibition of the calcineurin/NFAT pathway. Thus, modulation of endothelial PMCA4 activity might be exploited in clinic to design new anti-angiogenic approaches.